The CER3 wax biosynthetic gene from Arabidopsis thaliana is allelic to WAX2/YRE/FLP1

The cuticle coats the aerial organs of land plants and is composed of a cutin matrix embedded and overlayed with waxes. The Arabidopsis CER3 gene is important for cuticular wax biosynthesis and was reported to correspond to At5g02310 encoding an E3 ubiquitin ligase. Here, we demonstrate that CER3 is not At5g02310 and instead corresponds to WAX2/YRE/FLP1 (At5g57800), a gene of unknown function required for wax biosynthesis. CER3 protein has also been implicated in cutin production because strong cer3 alleles display organ fusions. Leaf cutin analysis of two cer3 alleles did not reveal significant differences in cutin load or composition, indicating that CER3 has no major role in leaf cutin formation.     

Reconstitution of Plant Alkane Biosynthesis in Yeast Demonstrates That Arabidopsis ECERIFERUM1 and ECERIFERUM3 Are Core Components of a Very-Long-Chain Alkane Synthesis Complex

In land plants, very-long-chain (VLC) alkanes are major components of cuticular waxes that cover aerial organs, mainly acting as a waterproof barrier to prevent nonstomatal water loss. Although thoroughly investigated, plant alkane synthesis remains largely undiscovered. The Arabidopsis thaliana ECERIFERUM1 (CER1) protein has been recognized as an essential element of wax alkane synthesis; nevertheless, its function remains elusive. In this study, a screen for CER1 physical interaction partners was performed. The screen revealed that CER1 interacts with the wax-associated protein ECERIFERUM3 (CER3) and endoplasmic reticulum-localized cytochrome b5 isoforms (CYTB5s). The functional relevance of these interactions was assayed through an iterative approach using yeast as a heterologous expression system. In a yeast strain manipulated to produce VLC acyl-CoAs, a strict CER1 and CER3 coexpression resulted in VLC alkane synthesis. The additional presence of CYTB5s was found to enhance CER1/CER3 alkane production. Site-directed mutagenesis showed that CER1 His clusters are essential for alkane synthesis, whereas those of CER3 are not, suggesting that CYTB5s are specific CER1 cofactors. Collectively, our study reports the identification of plant alkane synthesis enzymatic components and supports a new model for alkane production in which CER1 interacts with both CER3 and CYTB5 to catalyze the redox-dependent synthesis of VLC alkanes from VLC acyl-CoAs.     

The Arabidopsis cer26 mutant,like the cer2 mutant,is specifically affected in the very long chain fatty acid elongation process

Plant aerial organs are covered by cuticular waxes, which form a hydrophobic crystal layer that mainly serves as a waterproof barrier. Cuticular wax is a complex mixture of very long chain lipids deriving from fatty acids, predominantly of chain lengths from 26 to 34 carbons, which result from acyl‐CoA elongase activity. The biochemical mechanism of elongation is well characterized; however, little is known about the specific proteins involved in the elongation of compounds with more than 26 carbons available as precursors of wax synthesis. In this context, we characterized the three Arabidopsis genes of the CER2‐like family: CER2, CER26 and CER26‐like . Expression pattern analysis showed that the three genes are differentially expressed in an organ‐ and tissue‐specific manner. Using individual T–DNA insertion mutants, together with a cer2 cer26 double mutant, we characterized the specific impact of the inactivation of the different genes on cuticular waxes. In particular, whereas the cer2 mutation impaired the production of wax components longer than 28 carbons, the cer26 mutant was found to be affected in the production of wax components longer than 30 carbons. The analysis of the acyl‐CoA pool in the respective transgenic lines confirmed that inactivation of both genes specifically affects the fatty acid elongation process beyond 26 carbons. Furthermore, ectopic expression of CER26 in transgenic plants demonstrates that CER26 facilitates the elongation of the very long chain fatty acids of 30 carbons or more, with high tissular and substrate specificity.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

Cloning and characterization of CER2, an Arabidopsis gene that affects cuticular wax accumulation.

Cuticular waxes are complex mixtures of very long chain fatty acids and their derivatives that cover plant surfaces. Mutants of the ECERIFERUM2 (cer2) gene of Arabidopsis condition bright green stems and siliques, indicative of the relatively low abundance of the cuticular wax crystals that comprise the wax bloom on wild-type plants. We cloned the CER2 gene via chromosome walking. Three lines of evidence establish that the cloned sequence represents the CER2 gene: (1) this sequence is capable of complementing the cer2 mutant phenotype in transgenic plants; (2) the corresponding DNA sequence isolated from plants homozygous for the cer2-2 mutant allele contains a sequence polymorphism that generates a premature stop codon; and (3) the deduced CER2 protein sequence exhibits sequence similarity to that of a maize gene (glossy2) that also is involved in cuticular wax accumulation. The CER2 gene encodes a novel protein with a predicted mass of 47 kD. We studied the expression pattern of the CER2 gene by in situ hybridization and analysis of transgenic Arabidopsis plants carrying a CER2-beta-glucuronidase gene fusion that includes 1.0 kb immediately upstream of CER2 and 0.2 kb of CER2 coding sequences. These studies demonstrate that the CER2 gene is expressed in an organ- and tissue-specific manner; CER2 is expressed at high levels only in the epidermis of young siliques and stems. This finding is consistent with the visible phenotype associated with mutants of the CER2 gene. Hence, the 1.2-kb fragment of the CER2 gene used to construct the CER2-beta-glucuronidase gene fusion includes all of the genetic information required for the epidermis-specific accumulation of CER2 mRNA.     

ABC-type transporters and cuticle assembly: Linking function to polarity in epidermis cells

The aerial organs of plants are covered with a cuticle, a continuous layer overlaying the outermost cell walls of the epidermis. The cuticle is composed of two major classes of the lipid biopolymers: cutin and waxes, collectively termed cuticular lipids. Biosynthesis and transport of cuticular lipids occur predominantly in the epidermis cells. In the transport pathway, cuticular lipids are exported from their site of biosynthesis in the ER/plastid to the extracellular space through the plasma membrane and cell wall. Growing evidence suggests that ATP-binding cassette (ABC) transporters are implicated in transport of cuticular lipids across the plasma membrane of epidermal cells. The Arabidopsis ABC-type transporter protein CER5 (WBC12) was reported to act as a wax monomers transporter. In recent works, our group and others showed that a CER5-related protein, DESPERADO (DSO/WBC11), is required for cutin and wax monomers transport through the plasma membrane of Arabidopsis epidermis cells. Unlike the cer5 mutant, DSO loss-of-function had a profound effect on plant growth and development, particularly dwarfism, postgenital organ fusions, and altered epidermal cell differentiation. The partially overlapping function of CER5 and DSO and the fact that these proteins are half-size ABC transporters suggest that they might form a hetero-dimeric complex while transporting wax components. An intriguing observation was the polar localization of DSO in the distal part of epidermis cells. This polar expression might be explained by DSO localization within lipid rafts, specific plasma membrane microdomains which are associated with polar protein expression. In this review we suggest possible mechanisms for cuticular lipids transport and a link between DSO function and polar expression. Furthermore, we also discuss the subsequent transport of cuticular constituents through the hydrophobic cell wall and the possible involvement of lipid transfer proteins in this process.Key words: ABC transporter, cuticular lipids, polar expression, plasma membrane, epidermis     

Overexpression of Arabidopsis ECERIFERUM1 promotes wax very-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stresses

Land plant aerial organs are covered by a hydrophobic layer called the cuticle that serves as a waterproof barrier protecting plants against desiccation, ultraviolet radiation, and pathogens. Cuticle consists of a cutin matrix as well as cuticular waxes in which very-long-chain (VLC) alkanes are the major components, representing up to 70% of the total wax content in Arabidopsis (Arabidopsis thaliana) leaves. However, despite its major involvement in cuticle formation, the alkane-forming pathway is still largely unknown. To address this deficiency, we report here the characterization of the Arabidopsis ECERIFERUM1 (CER1) gene predicted to encode an enzyme involved in alkane biosynthesis. Analysis of CER1 expression showed that CER1 is specifically expressed in the epidermis of aerial organs and coexpressed with other genes of the alkane-forming pathway. Modification of CER1 expression in transgenic plants specifically affects VLC alkane biosynthesis: waxes of TDNA insertional mutant alleles are devoid of VLC alkanes and derivatives, whereas CER1 overexpression dramatically increases the production of the odd-carbon-numbered alkanes together with a substantial accumulation of iso-branched alkanes. We also showed that CER1 expression is induced by osmotic stresses and regulated by abscisic acid. Furthermore, CER1-overexpressing plants showed reduced cuticle permeability together with reduced soil water deficit susceptibility. However, CER1 overexpression increased susceptibility to bacterial and fungal pathogens. Taken together, these results demonstrate that CER1 controls alkane biosynthesis and is highly linked to responses to biotic and abiotic stresses.     

Cuticular waxes on eceriferum mutants of Arabidopsis thaliana

We present cuticular wax chemical profiles for the leaves and stems of Arabidopsis wildtype Landsberg erecta and eleven isogenic eceriferum mutants: cer5, cer10 to cer15, and cer17 to cer20. These cer mutants have wax profiles that are different from those of wildtype in chemical chain length distribution, amount per chemical class, and/or total wax load. Analyses of detailed leaf and stem wax profiles for these cer mutants have allowed us to place some of these mutants at specific steps in wax production. The cer13 gene is predicted to affect release of the 30 carbon fatty acid from the elongation complex or the reduction of C30 fatty acid to C30 aldehyde. The CER19 gene product is predicted to be involved in C28 to C30 fatty acyl-CoA elongation. The CER20 gene is predicted to affect the oxidation of C29 alkane to C29 secondary alcohol. Several predicted gene products affect only stem specific steps in the wax pathway.     

Characterization of Arabidopsis ABCG11/WBC11, an ATP binding cassette (ABC) transporter that is required for cuticular lipid secretion

ABCG11/WBC11, an ATP binding cassette (ABC) transporter from Arabidopsis thaliana, is a key component of the export pathway for cuticular lipids. Arabidopsis wbc11 T-DNA insertional knock-out mutants exhibited lipidic inclusions inside epidermal cells similar to the previously characterized wax transporter mutant cer5, with a similar strong reduction in the alkanes of surface waxes. Moreover, the wbc11 knock-out mutants also showed defects not present in cer5, including post-genital organ fusions, stunted growth and a reduction in cutin load on the plant surface. A mutant line previously isolated in a forward genetics screen, called permeable leaves 1 (pel1), was identified as an allele of ABCG11/WBC11. The double knock-out wbc11 cer5 exhibited the same morphological and biochemical phenotypes as the wbc11 knock-out. A YFP-WBC11 fusion protein rescued a T-DNA knock-out mutant and was localized to the plasma membrane. These results show that WBC11 functions in secretion of surface waxes, possibly by interacting with CER5. However, unlike ABCG12/CER5, ABCG11/WBC11 is important to the normal process of cutin formation.     

CUT1, an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme.

Land plants secrete a layer of wax onto their aerial surfaces that is essential for survival in a terrestrial environment. This wax is composed of long-chain, aliphatic hydrocarbons derived from very-long-chain fatty acids (VLCFAs). Using the Arabidopsis expressed sequence tag database, we have identified a gene, designated CUT1, that encodes a VLCFA condensing enzyme required for cuticular wax production. Sense suppression of CUT1 in transgenic Arabidopsis plants results in waxless (eceriferum) stems and siliques as well as conditional male sterility. Scanning electron microscopy revealed that this was a severe waxless phenotype, because stems of CUT1-suppressed plants were completely devoid of wax crystals. Furthermore, chemical analyses of waxless plants demonstrated that the stem wax load was reduced to 6 to 7% of wild-type levels. This value is lower than that reported for any of the known eceriferum mutants. The severe waxless phenotype resulted from the downregulation of both the decarbonylation and acyl reduction wax biosynthetic pathways. This result indicates that CUT1 is involved in the production of VLCFA precursors used for the synthesis of all stem wax components in Arabidopsis. In CUT1-suppressed plants, the C24 chain-length wax components predominate, suggesting that CUT1 is required for elongation of C24 VLCFAs. The unique wax composition of CUT1-suppressed plants together with the fact that the location of CUT1 on the genetic map did not coincide with any of the known ECERIFERUM loci suggest that we have identified a novel gene involved in wax biosynthesis. CUT1 is currently the only known gene with a clearly established function in wax production.     

An ABC transporter gene of Arabidopsis thaliana, AtWBC11, is involved in cuticle development and prevention of organ fusion

Cuticle, including wax and cutin, is the barrier covering plant aerial organs and protecting the inner tissues. The Arabidopsis thaliana ATP-binding cassette (ABC) transporter CER5 (AtWBC12) has been identified as a wax exporter. In agreement with the latest report of another wax exporter, AtWBC11, here we show that atwbc11 mutants displayed organ fusions and stunted growth, and became vulnerable to chlorophyll leaching and toluidine blue staining. Chemical analysis showed that wax and cutin monomers were both reduced in the atwbc11 mutant. AtWBC11 was widely expressed in aerial organs. Interestingly, we found that the expression was light dependent, and the phytohormone ABA up-regulated AtWBC11 expression. We also found that while the AtWBC11 promoter had a broad pattern of activity, the expression was converted to epidermis specific when the reporter gene was fused to AtWBC11 cDNA. Furthermore, RNA blot analysis supported epidermis-specific expression of AtWBC11. Our results support that AtWBC11 is involved in cuticle development.     

Alterations in CER6, a gene identical to CUT1, differentially affect long-chain lipid content on the surface of pollen and stems

Very long chain lipids contribute to the hydrophobic cuticle on the surface of all land plants and are an essential component of the extracellular pollen coat in the Brassicaceae. Mutations in Arabidopsis CER genes eliminate very long chain lipids from the cuticle surface and, in some cases, from the pollen coat. In Arabidopsis, the loss of pollen coat lipids can disrupt interactions with the stigma, inhibiting pollen hydration and causing sterility. We have positionally cloned CER6 and demonstrate that a wild-type copy complements the cer6-2 defect. In addition, we have identified a fertile, intragenic suppressor, cer6-2R, that partially restores pollen coat lipids but does not rescue the stem wax defect, suggesting an intriguing difference in the requirements for CER6 activity on stems and the pollen coat. Importantly, analysis of this suppressor demonstrates that low amounts of very long chain lipids are sufficient for pollen hydration and germination. The predicted CER6 amino acid sequence resembles that of fatty acid-condensing enzymes, consistent with its role in the production of epicuticular and pollen coat lipids >28 carbons long. DNA sequence analysis revealed the nature of the cer6-1, cer6-2, and cer6-2R mutations, and segregation analysis showed that CER6 is identical to CUT1, a cDNA previously mapped to a different chromosome arm. Instead, we have determined that a new gene, CER60, with a high degree of nucleotide and amino acid similarity to CER6, resides at the original CUT1 locus.     

Cloning and characterization of the WAX2 gene of Arabidopsis involved in cuticle membrane and wax production

Insertional mutagenesis of Arabidopsis ecotype C24 was used to identify a novel mutant, designated wax2, that had alterations in both cuticle membrane and cuticular waxes. Arabidopsis mutants with altered cuticle membrane have not been reported previously. Compared with the wild type, the cuticle membrane of wax2 stems weighed 20.2% less, and when viewed using electron microscopy, it was 36.4% thicker, less opaque, and structurally disorganized. The total wax amount on wax2 leaves and stems was reduced by >78% and showed proportional deficiencies in the aldehydes, alkanes, secondary alcohols, and ketones, with increased acids, primary alcohols, and esters. Besides altered cuticle membranes, wax2 displayed postgenital fusion between aerial organs (especially in flower buds), reduced fertility under low humidity, increased epidermal permeability, and a reduction in stomatal index on adaxial and abaxial leaf surfaces. Thus, wax2 reveals a potential role for the cuticle as a suppressor of postgenital fusion and epidermal diffusion and as a mediator of both fertility and the development of epidermal architecture (via effects on stomatal index). The cloned WAX2 gene (verified by three independent allelic insertion mutants with identical phenotypes) codes for a predicted 632-amino acid integral membrane protein with a molecular mass of 72.3 kD and a theoretical pI of 8.78. WAX2 has six transmembrane domains, a His-rich diiron binding region at the N-terminal region, and a large soluble C-terminal domain. The N-terminal portion of WAX2 is homologous with members of the sterol desaturase family, whereas the C terminus of WAX2 is most similar to members of the short-chain dehydrogenase/reductase family. WAX2 has 32% identity to CER1, a protein required for wax production but not for cuticle membrane production. Based on these analyses, we predict that WAX2 has a metabolic function associated with both cuticle membrane and wax synthesis. These studies provide new insight into the genetics and biochemistry of plant cuticle production and elucidate new associations between the cuticle and diverse aspects of plant development.     

Developmental and hormonal regulation of the arabidopsis CER2 gene that codes for a nuclear-localized protein required for the normal accumulation of cuticular waxes.

The previously cloned CER2 gene is required for the normal accumulation of cuticular waxes and encodes a novel protein. Earlier reports suggested that the CER2 protein is either a membrane-bound component of the fatty acid elongase complex or a regulatory protein. Cell fractionation and immunoblot analyses using polyclonal antibodies raised against a chemically synthesized peptide with a sequence based on the predicted CER2 protein sequence have demonstrated that the 47-kD CER2 protein is soluble and nuclear localized. These results are consistent with CER2 being a regulatory protein. Detailed studies of plants harboring a CER2 promoter/GUS transgene (CER2-GUS), in combination with immunoblot analyses, revealed that CER2 is expressed and the CER2 protein accumulates in a variety of organs and cell types. Expression is highest early in the development of these organs and is epidermis specific in most tissues. In agreement with the activity of the CER2 promoter in hypocotyls, cuticular wax accumulates on this organ in a CER2-dependent fashion. In leaves CER2 expression is confined to the guard cells, trichomes, and petioles. However, application of the cytokinin 6-benzylaminopurine induces ectopic expression of CER2-GUS in all cell types of leaves that emerge following treatment.